The present invention relates to methods for modulating vascularization particularly in a mammal. In one aspect, methods are provided for modulating vascularization that includes administrating to the mammal an effective amount of granulocyte macrophage-colony stimulating factor (GM-CSF). Further provided are methods for treating or detecting damaged blood vessels in the mammal. The invention has a wide spectrum of useful applications including inducing formation of new blood vessels in the mammal.
There is nearly universal recognition that blood vessels help supply oxygen and nutrients to living tissues. Blood vessels also facilitate removal of waste products. Blood vessels are renewed by a process termed xe2x80x9cangiogenesisxe2x80x9d. See generally Folkman and Shing, J. Biol. Chem. 267 (16), 10931-10934 (1992).
Angiogenesis is understood to be important for the well-being of most mammals. As an illustration, angiogenesis has been disclosed as being an essential process for reproduction, development and wound repair.
There have been reports that inappropriate angiogenesis can have severe consequences. For example, it has been disclosed that solid tumor growth is facilitated by vascularization. There is broad support for the concept that mammals must regulate angiogenesis extensively.
There has been much attention directed to understanding how angiogeneis is controlled. In particular, angiogenesis is believed to begin with the degradation of the basement membrane by proteases secreted from endothelial cells (EC) activated by mitogens, e.g., vascular endothelial growth factor (ie. VEGF-1), basic fibroblast growth factor (bFGF) and/or others. The cells migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space, then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of new basement membrane.
In adults, it has been disclosed that the proliferation rate of endothelial cells is typically low, compared to other cell types in the body. The turnover time of these cells can exceed one thousand days. Physiological exceptions in which angiogenesis results in rapid proliferation occurs under tight regulation are found in the female reproduction system and during wound healing. It has been reported that the rate of angiogenesis involves a change in the local equilibrium between positive and negative regulators of the growth of microvessels.
Abnormal angiogenesis is thought to occur when the body loses its control of angiogenesis, resulting in either excessive or insufficient blood vessel growth. For instance, conditions such as ulcers, strokes, and heart attacks may result from the absence of angiogenesis normally required for natural healing. In contrast, excessive blood vessel proliferation can facilitate tumor growth, blindness, psoriasis, rheumatoid arthritis, as well as other medical conditions.
The therapeutic implications of angiogenic growth factors were first described by Folkman and colleagues over two decades ago (Folkman, N. Engl. J. Med., 85:1182-1186 (1971)). Recent work has established the feasibility of using recombinant angiogenic growth factors, such as fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) and Baffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell growth factor (ECGF)(Pu, et al., J Surg Res, 54:575-83 (1993)), and vascular endothelial growth factor (VEGF-1) to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93:662-70 (1-994)).
The feasibility of using gene therapy to enhance angiogenesis has received recognition. For example, there have been reports that angiogenesis can facilitate treatment of ischemia in a rabbit model and in human clinical trials. Particular success has been achieved using VEGF-1 administered as a balloon gene delivery system. Successful transfer and sustained expression of the VEGF-1 gene in the vessel wall subsequently augmented neovascularization in the ischemic limb (Takeshita, et al., Laboratory Investigation, 75:487-502 (1996); Isner, et al., Lancet, 348:370 (1996)). In addition, it has been reported that direct intramuscular injection of DNA encoding VEGF-1 into ischemic tissue induces angiogenesis, providing the ischemic tissue with increased blood vessels (Tsurumi et al., Circulation, 94(12):3281-3290 (1996)).
Alternative methods for promoting angiogenesis are desirable for a number of reasons. For example, it is believed that native endothelial progenitor cell (EPC) number and/or viability decreases over time. Thus, in certain patient populations, e.g., the elderly, EPCs capable of responding to angiogenic proteins may be limited. Also, such patients may not respond well to conventional therapeutic approaches.
There have been reports that at least some of these problems can be reduced by administering isolated EPCs to patients and especially those undergoing treatment for ischemic disease. However, this suggestion is believed to be prohibitively expensive as it can require isolation and maintenance of patient cells. Moreover, handling of patient cells can pose a significant health risk to both the patient and attending personnel in some circumstances.
Granulocyte macrophage colony stimulating factor (GM-CSF) has been shown to exert a regulatory effect on granulocyte-committed progenitor cells to increase circulating granulocyte levels (Gasson, J. C., Blood 77:113 1 (1991). In particular, GM-CSF acts as a growth factor for granulocyte, monocyte and eosinophil progenitors.
Administration of GM-CSF to human and non-human primates results in increased numbers of circulating neutrophils, as well as eosinophils, monocytes and lymphocytes. Accordingly, GM-CSF is believed to be particularly useful in accelerating recovery from neutropenia in patients subjected to radiation or chemotherapy, or following bone marrow transplantation. In addition, although GM-CSF is less potent than other cytokines, e.g., FGF, in promoting EC proliferation, GM-CSF activates a fully migrating phenotype. (Bussolino, et al., J. Clin. Invent., 87:986 (1991).
Accordingly, it would be desirable to have methods for modulating vascularization in a mammal and especially a human patient. It would be particularly desirable to have methods that increase EPC mobilization and neovascularization (formation of new blood vessels) in the patient that do not require isolation of EPC cells.
The present invention generally relates to methods for modulating vascularization in a mammal. In one aspect, the invention provides methods for increasing vascularization that includes administrating to the mammal an effective amount of a vascularization modulating agent, such as granulocyte macrophage-colony stimulating factor (GM-CSF), VEGF, Steel factor (SLF, also known as Stem cell factor (SCF)), stromal cell-derived factor (SDF-1), granulocyte-colony stimulating factor (G-CSF), HGF, Angiopoietin-1, Angiopoietin-2, M-CSF, b-FGF, and FLT-3 ligand, and effective fragment thereof, or DNA coding for such vascularization modulating agents. Such materials have sometimes previously been described as xe2x80x9chematopoietic factors.xe2x80x9d and/or xe2x80x9chematopoietic proteins.xe2x80x9d Disclosure relating to these and other hematopoietic factors can be found in Kim, C. H. and Broxmeyer, H. E. (1998) Blood, 91:100; Turner, M. L. and Sweetenham, J. W., Br. J. Haematol. (1996) 94:592; Aiuuti, A. et al. (1997) J. Exp. Med. 185:111; Bleul, C. et al. (1996) J. Exp. Med. 184:1101; Sudo, Y. et al. (1 997) Blood, 89: 3166; as well as references disclosed therein. Prior to the present invention, it was not kown that GM-CSF or other hematopoietic factors could potentiate endothelial progenitor cells, or modulate neovascularization as described herein.
Alternatively, instead of the proteins themselves or effective fragments thereof, the DNA coding for the vascularization modulating agents can be administered to the site where neovascularization is desired, as further discussed below. The invention also relates to methods for treating or detecting damaged blood vessels in the mammal. The invention has many uses including preventing or reducing the severity of blood vessel damage associated with ischemia or related conditions.
We have now discovered that hematopoietic factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), modulate endothelial progenitor cell (EPC) mobilization and neovascularization (blood vessel formation). In particular, we have found that GM-CSF and other hematopoietic factors increase EPC mobilization and enhances neovascularization. This observation was surprising and unexpected in light of prior reports addressing GM-CSF activity in vitro and in vivo. Accordingly, this invention provides methods for using GM-CSF to promote EPC mobilization and to enhance neovascularization, especially in tissues in need of EPC mobilization and/or neovascularization.
In one aspect, the present invention provides a method for inducing neovascularization in a mammal. By the term xe2x80x9cinductionxe2x80x9d is meant at least enhancing EPC mobilization and also preferably facilitating formation of new blood vessels in the mammal. EPC mobilization is understood to mean a significant increase in the frequency and differentiation of EPCs as determined by assays disclosed herein. In one embodiment, the method includes administering to the mammal an effective amount of a vascularization modulating factor such as granulocyte macrophage-colony stimulating factor (GM-CSF), that is preferably sufficient to induce the neovascularization in the mammal. Preferably, that amount of GM-CSF is also capable of modulating and particularly increasing frequency of EPCs in the mammal. A variety of methods for detecting and quantifying neovascularization, EPC frequency, the effectiveness of vascularization modulating agents, and other parameters of blood vessel growth are discussed below and in the examples.
In a particular embodiment of the method, the enhancement in EPC mobilization and particularly the increase in frequency of the EPCs is at least about 20% and preferably from between 50% to 500% as determined by a standard EPC isolation assay. That assay generally detects and quantifies EPC enrichment and is described in detail below.
In another particular embodiment of the method, the amount of administered modulating agent is sufficient to enhance EPC mobilization and especially to increase EPC differentiation in the mammal. Methods for detecting and quantifying EPC differentiation include those specific methods described below. Preferably, the increase in EPC differentiation is at least about 20%, preferably between from about 100% to 1000%, more preferably between from about 200% to 800% as determined by a standard EPC culture assay discussed below. More preferably, that amount of administered modulating agent is additionally sufficient to increase EPC differentiation by about the stated percent amounts following tissue ischemia as determined in a standard hindlimb ischemia assay as discussed below.
In another particular embodiment of the method, the amount of vascularization modulating agent administered to the mammal is sufficient to increase blood vessel size in the mammal. Methods for determining parameters of blood vessel size, e.g., length and circumference, are known in the field and are discussed below. Preferably, the amount of administered modulating agent is sufficient to increase blood vessel length by at least about 5%, more preferably between from about 10% to 50%, even more preferably about 20%, as determined by a standard blood vessel length assay discussed below. Preferably, the amount of modulating agent administered to the mammal is also sufficient to increase blood vessel circumference or diameter by the stated percent amounts as determined by a standard blood vessel diameter assay. As will be discussed below, it will often be preferred to detect and quantify changes in blood vessel size using a standard cornea micropocket assay, although other suitable assays can be used as needed.
In another particular embodiment of the method, the amount of administered vascularization modulating agent is sufficient to increase neovascularization by at least about 5%, preferably from between about 50% to 300%, and more preferably from between about 100% to 200% as determined by the standard cornea micropocket assay. Methods for performing that assay are known in the field and include those specific methods described below. Additionally, preferred amounts of GM-CSF are sufficient to improve ischemic hindlimb blood pressure by at least about 5%, preferably between from about 10% to 50% as determined by standard methods for measuring the blood pressure of desired vessels. More specific methods for measuring blood pressure particularly with new or damaged vessels include techniques optimized to quantify vessel pressure in the mouse hindlimb assay discussed below.
In another particular embodiment of the method, the amount of administered vascularization modulating agent is sufficient to increase EPC bone marrow (BM) derived EPC incorporation into foci by at least about 20% as determined by a standard murine BM transplantation model. Preferably, the increase is between from about 50% to 400%, more preferably between from about 100% to 300% as determined by that standard model. More specific methods for determining the increase in EPC incorporation into foci are found in the discussion and Examples which follow.
The methods of this invention are suitable for modulating and especially inducing neovascularization in a variety of animals including mammals. The term xe2x80x9cmammalxe2x80x9d is used herein to refer to a warm blooded animal such as a rodent, rabbit, or a primate and especially a human patient. Specific rodents and primates of interest include those animals representing accepted models of human disease including the mouse, rat, rabbit, and monkey. Particular human patients of interest include those which have, are suspected of having, or will include ischemic tissue. That ischemic tissue can arise by nearly any means including a surgical manipulation or a medical condition. Ischemic tissue is often associated with an ischemic vascular disease such as those specific conditions and diseases discussed below.
As will become more apparent from the discussion and Examples which follow, methods of this invention are highly compatible and can be used in combination with established or experimental methods for modulating neovascularization. In one embodiment, the invention includes methods for modulating and particularly inducing neovascularization in a mammal in which an effective amount of vascularization modulating agent is co-administered with an amount of at least one angiogenic protein. In many settings, it is believed that co-administration of the vascularization modulating agent and the angiogenic protein can positively impact neovascularization in the mammal, e.g., by providing additive or synergistic effects. A preferred angiogenic protein is a recognized endothelial cell mitogen such as those specific proteins discussed below. Methods for co-administering the vascularization modulating agent and the angiogenic protein are described below and will generally vary according to intended use.
The present invention also provides methods for preventing or reducing the severity of blood vessel damage in a mammal such as a human patient in need of such treatment. In one embodiment, the method includes administering to the mammal an effective amount of vascularization modulating agent such as GM-CSF. At about the same time or subsequent to that administration, the mammal is exposed to conditions conducive to damaging the blood vessels. Alternatively, administration of the vascularization modulating agent can occur after exposure to the conditions to reduce or block damage to the blood vessels. As discussed, many conditions are known to induce ischemic tissue in mammals which conditions can be particularly conducive to damaging blood vessels, e.g, invasive manipulations such as surgery, grafting, or angioplasty; infection or ischemia. Additional conditions and methods for administering the vascularization modulating agent are discussed below.
Preferred amounts of the vascularization modulating agent to use in the methods are sufficient to prevent or reduce the severity of the blood vessel damage in the mammal. Particular amounts of GM-CSF have already been mentioned above and include administration of an effective amount of GM-CSF sufficient to induce neovascularization in the mammal. Illustrative methods for quantifying an effective amount of vascularization modulating agents are discussed throughout this disclosure including the discussion and Examples which follow.
The present invention also provides methods for treating ischemic tissue and especially injured blood vessels in that tissue. Preferably, the method is conducted with a mammal and especially a human patient in need of such treatment. In one embodiment, the method includes as least one and preferably all of the following steps:
a) isolating endothelial progenitor cells (EPCs) from the mammal,
b) contacting the isolated EPCs with an effective amount of at least one factor sufficient to induce proliferation of the EPCs; and
c) administering the proliferated EPCs to the mammal in an amount sufficient to treat the injured blood vessel.
In a particular embodiment of the method, the factor is an angiogenic protein including those cytokines known to induce EPC proliferation especially in vitro. Illustrative factors and markers for detecting EPCs are discussed below. In one embodiment of the method, the blood vessel (or more than one blood vessel) can be injured by nearly any known means including trauma or an invasive manipulation such as implementation of balloon angioplasty or deployment of a stent or catheter. A particular stent is an endovascular stent. Alternatively, the vascular injury can be organic and derived from a pre-existing or on-going medical condition.
In another particular embodiment of the method, the vascularization modulating agent is administered to the mammal and especially the human patient alone or in combination (co-administered) with at least one angiogenic protein (or effective fragment thereof) such as those discussed below.
Additionally provided by this invention are methods for detecting presence of tissue damage in a mammal and especially a human patient. In one embodiment, the method includes contacting the mammal with a detectably-labeled population of EPCs; and detecting the detectably-labeled cells at or near the site of the tissue damage in the mammal. In this example, the EPCs can be harvested and optionally monitored or expanded in vitro by nearly any acceptable route including those specific methods discussed herein. The EPCs can be administered to the mammal by one or a combination of different approaches with intravenous injection being a preferred route for most applications. Methods for detectably-labeling cells are known in the field and include immunological or radioactive tagging as well as specific recombinant methods disclosed below.
In a particular embodiment of the method, the detectably-labeled EPCs can be used to xe2x80x9chome-inxe2x80x9d to a site of vascular damage, thereby providing a minimally invasive means of visualizing that site even when it is quite small. The detectably-labeled EPCs can be visualized by a variety of methods well-known in this field including those using tomography, magnetic resonance imaging, or related approaches.
In another embodiment of the method, the tissue damage is facilitated by ischemia, particularly an ischemic vascular disease such as those specifically mentioned below.
Also provided by this invention are methods for modulating the mobilization of EPCs which methods include administering to the mammal an effective amount of at least one hematopoietic factor. Preferred are methods that enhance EPC mobilization as determined by any suitable assay disclosed herein. For example, in a particular embodiment of the method, the enhancement in EPC mobilization and particulary the increase in frequency of the EPCs is at least about 20% and preferably from between 50% to 500% as determined by a standard EPC isolation assay.
In another particular embodiment of the method, the amount of administered hematopoietic factor is sufficient to enhance EPC mobilization and especially to increase EPC differentiation in the mammal. Methods for detecting and quantifying EPC differentiation include those specific methods described below. Preferably, the increase in EPC differentiation is at least about 20%, preferably between from about 100% to 1000%, more preferably between from about 200% to 800% as determined by a standard EPC culture assay discussed below. More preferably, that amount of administered hematopoietic factor is additionally sufficient to increase EPC differentiation by about the stated percent amounts following tissue ischemia as determined in a standard hindlimb ischemia assay as discussed below.
As discussed, it has been found that EPC mobilization facilitates significant induction of neovascularization in mammals. Thus, methods that modulate EPC mobilization and particularly enhance same can be used to induce neovascularization in the mammal and especially a human patient in need of such treatment. Methods of this invention which facilitate EPC mobilization including those employing at least one hematopoietic factor which use can be alone or in combination with other methods disclosed herein including those in which an effective amount of vascularization modulating agent is administered to the mammal alone or in combination (co-administered) with at least one angiogenic protein.
In particular, the invention provides methods for inducing neovascularization in a mammal and especially a human patient in need of such treatment which methods include administering to the mammal an effective amount of at least one vascularization modulating agent, preferably one vascularization modulating agent, which amount is sufficient to induce neovascularization in the mammal. That neovascularization can be detected and quantified if desired by the standard assays disclosed herein including the mouse cornea micropocket assay and blood vessel size assays. Preferred methods will enhance neovascularization in the mammal by the stated percent ranges discussed previously.
In one embodiment of the method, the effective amount of the vascularization modulating agent(s) is co-administered in combination with at least one angiogenic protein, preferably one angiogenic protein. The vascularization modulating agent can be administered to the mammal and especially a human patient in need of such treatment in conjunction with, subsequent to, or following administration of the angiogenic or other protein.
The invention also provides a pharmaceutical product that is preferably formulated to modulate and especially to induce neovascularization in a mammal. In a preferred embodiment, the product is provided sterile and optionally includes an effective amount of GM-CSF and optionally at least one angiogenic protein. In a particular embodiment, the product includes isolated endothelial progenitor cells (EPCs) in a formulation that is preferably physiologically acceptable to a mammal and particularly a human patient in need of the EPCs. Alternatively, the product can include a nucleic acid that encodes the GM-CSF and/or the angiogenic protein.
Also provided by this invention are kits preferably formulated for in vivo and particularly systemic introduction of isolated EPCs. In one embodiment, the kit includes isolated EPCs and optionally at least one angiogenic protein or nucleic acid encoding same. Preferred is a kit that optionally includes a pharmacologically acceptable carrier solution, nucleic acid or mitogen, means for delivering the EPCs and directions for using the kit. Acceptable means for delivering the EPCs are known in the field and include effective delivery by stent, catheter, syringe or related means.
Other aspects of the invention are disclosed infra.